RNAscope The Journal of Molecular Diagnostics Fay Wang, John Flanagan, Nan Su, Li-Chong Wang, Son Bui, Allissa Nielson, Xingyong Wu, Hong-Thuy Vo, Xiao-Jun Ma, Yuling Luo  The Journal of Molecular Diagnostics  Volume 14, Issue 1, Pages 22-29 (January 2012) DOI: 10.1016/j.jmoldx.2011.08.002 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of the RNAscope assay procedure. In step 1, cells or tissues are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using an epifluorescent microscope (for fluorescent label) or standard bright-field microscope (for enzyme label). The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Validation of RNAscope. A: HeLa cells were hybridized with either the full set of probes to 18S rRNA, the left half of the set, or the right half of the set (as shown in the schematic along the top). A no-probe control was performed in parallel as an indicator of background staining. Cells were counterstained with DAPI (blue), which masks nucleolar 18S RNA. Scale bar = 10 μm. B: HCV-uninfected (left) or HCV-infected (right) HuH-7 cells were hybridized with probe sets to HCV mRNA (green). Cells were costained with 18S rRNA target probes (red) as an internal control for assay success. Nuclei were counterstained with DAPI (blue). Original magnification, ×40. C: HeLa cells were hybridized with probes to β-actin, RPLP0 (60S acidic ribosomal protein P0), PPIB (peptidylprolyl isomerase B), and HPRT-1 (hypoxanthine phosphoribosyltransferase 1) in multiplex fluorescence format. Nuclei were counterstained with DAPI. Original magnification, ×40. The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Single RNA molecule detection. A: HER2 genomic DNA in HeLa and SK-BR-3 cells was detected using the RNAscope probes and signal amplification system. Nuclei were costained for IL-8 for diploid genome Original magnification, ×40. B: HER2 mRNA detection in HeLa cells using the same probes and signal amplification system. A probe set to 18S rRNA was used as internal control for RNA detection. HER2 mRNA dots were counted; mean dots per cell (±95% confidence limit) are indicated. Nuclei were counterstained with DAPI (blue). Original magnification, ×40. C: HER2 mRNA copies per cell in HeLa cells determined by QuantiGene 2.0 (QG2) using a standard curve from in vitro transcribed RNA. Mean copies per cell (±95% confidence limit) are indicated (green dot), as calculated from triplicate measurements using linear regression. The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 RNAscope detection of RNA in FFPE tumor tissues. A: Chromogenic staining (DAB) of primary tumor tissues (breast, lung, and prostate) hybridized with either probes to ubiquitin C (UBC) or probes against the bacterial gene dapB as negative control. Nuclei were counterstained with hematoxylin. Original magnification, ×40. B: Fluorescent detection of low-copy transcripts in FFPE samples. Breast tumor tissue section was hybridized with either no probes or with Alexa Fluor 488-labeled probe sets (green) to HPRT1 or POLR2A. Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm. The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Detection of Ig κ chain expression in B lymphocytes in FFPE human tonsil tissue. κ light chain mRNA transcripts were stained using RNAscope or a commercial non-radioisotopic RNA ISH kit. For RNAscope, a negative control (bacterial gene dapB) was also included. The dotted line outlines the mantle zone. Original magnification, ×40. The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Multiplex fluorescence detection of uPA and PAI mRNAs in breast cancer. Merged pseudo-colored image of a metastatic breast cancer tissue section stained with probes specific to cytokeratins [PanCK (CK-8, CK-18, and CK-19), labeled with Alexa Fluor 647], uPA (labeled with Alexa Fluor 546), and PAI-1 (labeled with Alexa Fluor 488). Both uPA expression (arrowhead and right inset) and coexpression with PAI-1 (arrow and left inset) were detected. Nuclei were counterstained with DAPI (blue). Original magnification, ×40. The Journal of Molecular Diagnostics 2012 14, 22-29DOI: (10.1016/j.jmoldx.2011.08.002) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions